The RNA regulatory controls that dictate the outcome of RNA virus infection are not fully understood. In particular, the post-transcriptional RNA modification N6-methyladenosine (m6A) is a potent and dynamic modulator of RNA function. However, little is known about the biological significance of this modification or how it regulates systems that perturb cellular homeostasis, such as RNA virus infection. This project aims to determine how m6A regulates the replication of hepatitis C virus (HCV), a positive strand RNA virus of the Flaviviridae family. Our central hypothesis is that m6A acts directly on the HCV RNA genome to regulate the fate of the RNA for the different stages of the viral life cycle. The rationale for the proposed research is that by understanding how m6A regulates RNA virus replication, we can manipulate and target m6A-targeted processes to design new and innovative approaches for the prevention and treatment of RNA virus infection. Guided by our preliminary data, our hypothesis will be tested by pursuing these three specific aims: 1) Identify the sites of m6A within the HCV RNA genome at single nucleotide resolution, 2) define the mechanism by which m6A regulates HCV RNA replication, 3) determine the mechanism by which m6A-binding proteins regulate HCV assembly. In the first aim, we will define the kinetics and sites of adenosine methylation on the HCV RNA genome at single nucleotide resolution by using m6A individual-nucleotide-resolution cross-linking and immunoprecipitation and direct RNA sequencing. For the second aim, we will use viral mutants with inactivated m6A sites and genetic depletion of the m6A methyltransferases and m6A-demethylase to identify the mechanism by which m6A affects HCV RNA replication. In the third aim, we will determine the mechanism by which the m6A-binding proteins differentially regulate HCV assembly by defining where they bind to the HCV RNA genome using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation and by identifying the functional domains of these proteins. Taken together, this approach will define how direct modification of the HCV RNA genome by m6A regulates the life cycle of HCV, thereby identifying and characterizing a novel RNA regulatory control to viral infection. Ultimately, a detailed understanding of how m6A affects HCV infection will uncover novel strategies to develop antiviral therapies to target this RNA regulatory control that is exploited by RNA viruses for their replication.